Spectinomycin, an inhibitor of plastid protein synthesis, can be used to mark specific cell layers in the shoot meristem of Brassica napus. Pale yellow‐green (YG) plants resulting from spectinomycin‐treatment can be propagated indefinitely in vitro. Microscopic examination showed that YG‐plants result from inactivation of plastids in the L2 and L3 layers and are composed of a pale green epidermis covering a white mesophyll layer. Epidermal cells of YG and normal green plants are similar and contain 10–20 small pale green plastids. YG plants are equivalent to periclinal chimeras with the important distinction that there is no genotypic difference between the white and green cell layers. Periclinal divisions of epidermal cells take place at all stages of leaf development to produce invaginations of green mesophyll located in sectors of widely varying sizes. A periclinal division rate of 1 in 3000–4000 anticlinal divisions for the adaxial epidermis, was 2–3‐fold higher than that estimated for the abaxial epidermis. Analysis of white and green mesophyll showed that chloroplasts are essential for palisade cell differentiation and this requirement is cell‐autonomous. Stable marking of cell lineages with spectinomycin is simple, rapid and reveals the requirement for functional plastids in cellular differentiation.